Synthesis of N-Benzylideneaniline by Schiff base reaction using Kinnow peel powder as Green catalyst and comparative study of derivatives through ANOVA techniques

The cheap and easy availability of the Kinnow peel waste has reported various applications due to presence of multifunctional groups. Therefore, in present study we explored its application to synthesize N-Benzylideneaniline and its derivatives based on Schiff base reaction. Kinnow peel powder is characterized by FTIR, TEM, SEM, XRD, EDX, and TGA for functional groups, morphology, surface, elements and thermal stability. Benzaldehyde, aniline, and their derivatives such as 4-methyl benzaldehyde, 4-hydroxy benzaldehyde, 4-methoxy benzaldehyde, and 4-methoxy aniline have been used to compare the efficacy of the Schiff base reaction using analysis of variance (ANOVA) and it has been observed that combination of Aniline and benzaldehyde for Schiff base reaction provided 85% yield of relative product.

www.nature.com/scientificreports/ such as catalyst developed from leftover pulp of Citrus limetta (Mausambi) which was waste material 41,42 . Therefore, it is necessary to develop novel biomass catalyst that will be more stable and reliable for many applications. Hugo Schiff reported the Schiff base by condensation reaction between aldehyde and amine in 1864 43 . These compounds contains azomethine group (-HC=N-) and behaves like Flexi-dentate ligand. Schiff base have wide applications in many types of biological activities like antibacterial 44 , antitumor 45 , anti-oxidant 46 , anti-inflammatory activity 47 , antifungal 48 and industrial applications. Schiff base ligand is generally used in the development of inorganic chemistry, co-ordination chemistry because they are able to generate complex with metal ions. Some of the Schiff base shows good catalytic performance at high temperature 49 . Synthesis of Schiff base is catalyzed by various type of chemical catalyst. However, these catalysts may be harmful for environment as well as for human being. To overcome these issues, some eco-friendly catalyst developed and reported.
Due to the high importance of eco-friendly catalysis in the Schiff Base reaction and potential of cost effectively, available fruit peel experiments have been designed to investigate its application as a catalyst in the present study. This is very first report where Kinnow peel powder is used as organic catalyst for Schiff base reaction between aniline and benzaldehyde.

Experimental methodology
Materials. Waste peels of Kinnow mandarin is selected and used as a catalyst in this study. Kinnow peels are collected from local fruit stalls in Jaipur, Rajasthan. Bruker Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), Thermogravimetric analysis (TGA), Energy dispersion X-ray spectroscopy (EDS), X-ray diffraction (XRD), Transmission emission spectroscopy (TEM), and Thin layer chromatography (TLC) are performed for characterization of Kinnow peel powder. All chemicals and solvents are used without any purification: DMSO, DCM, acetonitrile, pet-ether, diethyl ether, aniline, benzaldehyde, 95% ethanol, and distilled water.
Preparation of the catalyst. Kinnow peel wastes are washed with di-ionized water to remove dirt particles and cleaned well. The peel is dried in an oven for 24 h at 70 ºC to remove moisture content. The dried peel waste is converted into powder form and then the powder is stored for further experiment (Fig. 1).
Preparation of Schiff base and their derivatives. 10 mg of Kinnow peel powder is added in reaction mixture of 1 mmol of Benzaldehyde and 1 mmol of Aniline in a test tube and allowed to stir it for 3 min on the magnetic stirrer at room temperature. The performance of the reaction is checked by the TLC plates with mobile phase [9:1 ratio of hexane and ethyl acetate] and after the completion of catalytic reaction the desired product is recrystallized by ethanol. Similar method is used for Schiff base reaction between derivatives of aniline and benzaldehyde.

Results and discussion
Herein, synthesis and to obtained high yield of the Schiff base product with green catalyst (organic material) is developed. This is achieved by adopting the green method for Schiff base reaction between benzaldehyde and aniline with Kinnow peel powder. It is observed that the desired product N-Benzylideneaniline is formed with 85% yield in 3 min (Scheme 1).
Optimization for Schiff base reaction:. The Schiff base reaction in between benzaldehyde and aniline is performed with various solvents (Table 1). From Table 1 it is clear that, 72% yield of relative product is observed with DCM (Table 1, entry 1), while DMSO provides only 70% yield (Table 1, entry 2). We also used diethyl ether, pet ether, acetonitrile for Schiff base reaction and 65-75% yield of relative products was found respectively  www.nature.com/scientificreports/ (Table 1, entry 3-5). We also investigated the effect of catalyst loading, it is observed that 10 mg catalyst provides best results (Table 1, entry 7) but without catalyst Schiff base reaction provides only 48% yield of relative product ( Table 1, entry 9). The bare component also provides moderate yield (60%) of Schiff base product (Table 1, entry  10). Therefore, Kinnow peel powder is high stable and eco-friendly catalyst for synthesis of Schiff base and providing best results under mild reaction conditions. Derivative of N-Benzylideneaniline also synthesized using same experimental procedure. The different derivatives such as 4-methyl benzaldehyde, 4-hydroxy benzaldehye, 4-methoxy benzaldehyde, and 4-methoxy aniline are used for Schiff base reaction by following optimized reaction conditions. Further more, it has been also observed that derivatives of aniline and benzaldehyde also affect the yield of relative Schiff base products. After that to confirm the yield of respective reaction, each derivative reaction of Schiff base put on five times and observed the result (As shown in Table 2) (Scheme 2).
The total number of sampled observation of the yield of six derivatives are not the same, therefore the difference among the effect of the derivatives to obtain by considering the following hypothesis. www.nature.com/scientificreports/ The null hypothesis (H 0 ): is that there is no difference among the effect of six derivatives on the yield of the product against the alternative hypothesis (H 1) : is that there is a difference among the effect of six derivatives on the yield of the product.
To test the above null hypothesis one-way analysis of variance (ANOVA) technique is used. Before applying the ANOVA technique, the assumption for homogeneity of variances is tested through Levene's test. For analysis of the data the software SPSS is used. From SPSS following Descriptive statistics is obtained ( Table 3).
The analysis of variance table for testing the difference among the derivatives on the yield is given below ( Table 4).
From the above table, it is concluded that there is a difference of the yield among the six derivatives. Since, there is a difference among six derivatives then a multiple comparison test that is a Tukey test is applied to find out the significant difference between any two derivatives.
From the Table 5 it is concluded that Aniline + Benzaldehyde has significant difference among these four derivatives, Aniline + 4-methyl benzaldehyde, Aniline + 4-hydroxybenzaldehyde, Aniline + 4-methoxy benzaldehyde, and 4-methoxy aniline + 4-methyl benzaldehyde. Aniline + benzaldehyde is not significantly different with 4-methoxy aniline + benzaldehyde. So on the basis of descriptive statistics the average yield of the product from the derivative Aniline + Benzaldehyde is more than 4-methoxy aniline + benzaldehyde. Therefore, the Aniline + Benzaldehyde has significant effect on the yield of the product.

IR spectrum of fresh and recovered Kinnow peel powder. IR spectrum of fresh Kinnow peel powder
and recovered Kinnow peel powder is compared (Fig. 2), which shows a broad peak at 3325 cm -1 and 3328 cm -1 in the high-frequency area attributed to the stretching mode of the O-H bond, which reveals the presence of hydroxyl groups in both Kinnow peel powder. The C-H stretching observed at 2920 cm -1 and 2918 cm -1 . The bands observed at 1718 cm -1 and 1720 cm -1 are assigned to the carboxyl group. The sharp peak found at 1605 cm -1 and 1603 cm -1 is a resonance peak which assigned to C = C (aromatic ring). The peak at 1419 cm -1 and 1406 cm -1 denotes COObond and the peak at 1093 cm -1 and 1094 cm -1 corresponds to the vibrational mode of the C-O group.   www.nature.com/scientificreports/ TEM. The TEM is used to determine the morphology of Kinnow peel powder. TEM images confirmed that the samples' particles are spherical having diameter of > 100 nm and rod shaped with > 50 nm in length (Fig. 3) and that the particles are largely agglomerated at 200 nm. The larger and variable sizes of Kinnow peel powder particles are visible in the TEM images.
SEM-EDX analysis. The scanning electron microscopy (SEM) has been used to study the morphology of Kinnow peel powder particles. Figure 4 shows the spectra obtained in SEM using EDX of the particle core. SEM spectra show the irregular particles with heterogeneous morphology. The size of the particle is 0.95 μm (calculated by imageJ). Figure 5 presents the result of EDX analysis for the cracked surface of Kinnow peel powder's particle after autoclave. It is evident that oxygen (O) and Ca are found as major (93.9%) and minor (6.1%) elements in the sample.
TGA analysis. Thermogravimetric analysis (TGA) is used to confirm Kinnow peel powder's thermal stability throughout a temperature range of 10 °C to 800 °C. As seen in Fig. 6. The removal of the chemisorbed and physisorbed solvent over the Kinnow peel powder's surface was clearly responsible for the weight loss below 200 °C. The huge weight loss has been observed in the temperature range of 230 °C to 510 °C. www.nature.com/scientificreports/ X-ray diffraction (XRD) of Kinnow peel powder. Figure 7 shows the X-ray diffraction (XRD) of Kinnow peel powder with key diffractions at 2θ = 15°, 19.7°, and 21° and it is observed with Cu Kɑ (λ = 1.5405 Å) radiation in the 2θ range from 10° to 90°. The activity of the catalyst (Kinnow peel powder), is compared with the reported methods for Schiff base reaction and the data shown in Table 6. From the data, it is clearly shows that the high efficiency of the present work while comparing on the literature reported catalysts for the Schiff base reaction.      www.nature.com/scientificreports/

Conclusion
N-Benzalideneaniline and their derivatives are synthesized by using benzaldehyde and aniline derivatives where Kinnow peel powder has been used as a catalyst. This reaction resulted into 85% to 78% yield in neat condition.
Comparisons of the yield of the six derivatives have also been done and comparative study done by ANOVA technique.